Background: Bad obstetric outcomes are considered as major health problem with social and medical impact on society, with multiple etiology.

Study Design: Descriptive Case Control Study

Aim: to evaluate the role of HPV-16 and HPV-18 in pregnancy loss in women in Kirkuk, Iraq.

Materials and Methods: The study included 60 women with BOH, of them 20 were pregnant and 20 were non-pregnant at time of enrolment in the study. In addition, 20 women with inevitable abortion were included in the study. Also the study included 28 women with normal pregnancy as control and 14 of them were pregnant and 14 were non-pregnant. Serological study carried out to determine HPV -16 IgG, and HPV -18 IgG antibodies using ELISA kits.

Results: the overall HPV seroprevalence was 3.4% for both HPV-16 and HPV-18. In women with BOH serprevalence was 7.5% and 2.5% for HPV-16 and HPV-18 respectively. While in control [normal pregnancy] none of the tested sera show a positive result. In women with inevitable abortion, HPV-18 detected in 10% of the tested samples. HPV-16 was with higher seroprevalence in non-pregnant (5.9%) as compared to pregnant (2.9%) women, however, not reach a significant level.

Conclusions: Although, HPV-16 and HPV-18 infections were detected in low rate in women with BOH and not in women with normal pregnancy, however, a large scale study warranted.

Human Papilloma virus is one of the tumor viruses and studies suggested a link between some HPV types and development of tumors such as oropharyngeal [1], and cervical cancer [2]. Bad obstetric outcome is one of the major social problems in Iraqi population with a wide range of etiology. Infections are implicated as an important cause of bad pregnancy outcomes [3,4]. Recent studies in Iraq suggest that Toxoplasma gondi, Cytomegalovirus, Rubella virus, and Herpes Simplex Virus infections were with positive association with BOH [5-14]. In addition, abnormal immunological responses may play a role in the pathogenesis of bad pregnancy outcomes [15,16].
Human papillomavirus is viral infection that may affect the pregnancy outcome [17]. Forty-eight studies provided HPV- type specific prevalence data in women with normal cytology. The five most common types of HPV were HPV 16 (2.5%), HPV 18 (0.9%), HPV 31 (0.7%), HPV 58 (0.6%) and HPV 52 (0.6%). The authors estimated that the overall burden of HPV infection from the age-specific adjusted prevalence in cytological normal women to be 291 million women infected with HPV of whom 23.3% were estimated to be infected with HPV 16 and 8.5% with HPV 18 [18]. BOH is still represent a medical and social problem and may microbiological infections reported to be associated with pregnancy loss [4].

Previous studies [19-29] outlining the prevalence of maternal HPV was identified. The seroprevalence of HPV-16 was with a range of 1.3% in China [22] to 97.71% in China [23].
While, HPV-18 seroprevalence was with a range of 0.5% in
China [22] to 27.6% in Brazil [25]. Since we don’t find a study in
literature that evaluates the association between HPV infection
and bad pregnancy outcomes. Thus this study was conducted to
clarify the risk assessment of HPV infection in women with bad
obstetric history.

The study design is a Descriptive Case Control Study and
was performed in Kirkuk General Hospital. The study proposal
was approved by Tikrit University College of Science ethical
committee and Kirkuk Health Authority Research Committee.
Informed consent taken from each woman included in the study.

The study population is women with childbearing age. Study
population was recruited from Kirkuk General Hospital. 88
women with age range from 14 to 48 were included in the study,
of them 60 with history of bad pregnancy outcomes and 28
women with normal pregnancy as control. The study included
60 women with BOH, of them 20 were pregnant and 20 were
non-pregnant at time of enrolment in the study. In addition, 20
women with inevitable abortion were included in the study. Also
the study included 28 women with normal pregnancy as control
and 14 of them were pregnant and 14 were non-pregnant. For
serological analysis, 5-10 mL of venous blood was collected in
a sterile container with strict aseptic precautions from each
study subject. The serum was separated and stored in numbered
aliquots at -20°C till assayed. All the serum samples collected
from the study and control groups were tested for HPV-16 and
HPV-18 IgG using commercially- available (ELISA) kits. The
results read by a Microwell reader and compared in a parallel
manner with controls; optical density read at 450 nm on an
ELISA reader.

All recruited women were subject for clinical examination and
laboratory investigations were carried out for the study subjects
to exclude other causes of foetal wastage, such as hypertension,
diabetes mellitus, syphilis, Rh (rhesus) incompatibility, physical
causes of abortion, and consanguinity. Subjects with known
causes of foetal wastage were excluded from the study. All of
them were interviewed to ascertain age, medical and obstetric
information.

The proportion and the mean value were computed in
appropriate situations. The data analyzed using the SPSS
(Version 16). The study finding data were presented as mean and
student t test and ANOVA were used to determine significance of
differences between groups.

The overall HPV seroprevalence in our study population
was 3.4% for both HPV 16 and HPV 18. In women with BOH
seroprevalence was 7.5% and 2.5% for HPV 16 and HPV 18
respectively, as shown in (Table 1) while in the control group
(normal pregnancy) none of the tested sera show a positive result.
In women with inevitable abortion, HPV 18 detected in 10% of
the tested samples.HPV 16 was with higher seroprevalence in
non pregnant (5.9%) as compared to pregnant (2.9%) women,
however, did not reach a significant level, as shown in (Table 2).
HPV 16 seroprevalence was 12% in women with age of 14-29
years, while all were negative in the control group as shown in
(Table 3). However, HPV 18 was seropositive in 8% of women in
the age of 14-29 years, and in 2.8% in women with age of 30-48 years, but the difference was not significant. There was no significant difference in HPV 16 and HPV 18 seroprevalence between
control and patients for age groups, as shown in (Table 3).

In the present study HPV 16 and HPV 18 seroprevalence
was 3.4% for each (3/88). In addition, HPV16 seroprevalence
was higher in women with BOH (7.5%) as compared to those
with normal pregnancy outcome (0%). However, this difference
was not statistically significant. Furthermore, all women with
inevitable abortion were negative for IgG HPV 16, while IgG HPV
18 was positive in 10% of women with inevitable abortion.

The present study seroprevalence of HPV 16 IgG in women
with BOH (7.5%) was higher than that reported for other
geographical areas using PCR technique, such as in China (1.3%)
[22], Brazil (5.3%) [25] and Iran (5.8%) [19]. However, our
finding was lower than that reported for Iran (10%, 73.9%)
[28, 29], West Africa (17.6%) [27], Egypt (20%) [20], Morocco
(42.5%) [21], Brazil (53.3%) [25], Saudi Arabia (71%) [24] and
China (97.7%) [23].Thus the prevalence of HPV16 varies in
different geographical areas and there may be wide a variation
in its prevalence within the same country. For example, in China
one research group reported the prevalence rate of 1.3%, while
other study reported the prevalence of 97.7% [23]. In addition,
the same pattern was reported for the Iran [28,29].

HPV 18 seroprevalence in women with BOH was 2.5%,
which was higher than the prevalence reported for China
(0.5%) [22] and Brazil (1.3%) [26]. However, the present study
seroprevalence was lower than that reported for Iran (4-11.6%)
[28], Egypt (10%) [20], China (14.2%) [23] West Africa (14.8%)
[27], Saudi Arabia (15%) [24] and Brazil (27.6%) (476). The
HPV 18 seroprevalence (2.5%) as this study indicated was lower
than that of HPV 16 seroprevalence (7.5%). This finding was
consistent to the studies reported globally which indicated a
high prevalence of HPV 16 as compared to HPV 18.

In addition, HPV 18 seroprevalence was higher in women
with inevitable abortion (10%) as compared to HPV 16
seroprevalence (0%). There was no reported data in literature
to compare with. However, this finding may be influenced by
the small size sample. Furthermore, both HPV 16 and HPV 18
seroprevalence was more in BOH women with age of <30 years,
which may be a reflection of sexual activity. However, OR did not
confirm an association between HPV 16 and HPV 18 infection
age of < 30 years. Ampuhl et al. [30] in a recent review found that there was an association between HPV infection and
spontaneous abortion or preterm delivery. Other study reported
that HPV infection was associated with preterm rupture of
membrane [31]. Genital HPV infection is the most common
sexually transmitted infection in the world although detection
of HPV types by polymerase chain reaction (PCR) assays varies
greatly with age and by geography. Clifford et al [18] in a study
that included women selected randomly from eleven countries
found that age standardized prevalence varied nearly 20 times
between different populations. The lower prevalence of 1.4% in
Spain, while the higher prevalence of 25.6% in Nigeria. In addition,
the overall, age standardized HPV prevalence was five times
higher in sub-Saharan Africa than in Europe with intermediate
prevalence in South America and Asia. Furthermore, HPV - 16
was the commonest type in all regions except in sub-Saharan
Africa, where HPV-35 was equally as common. Sanjose, et al.
[32] in a large scale meta-analysis study that included 157 879
women with normal cytology found that HPV global prevalence
was 10.4% and was less in developed countries (10.6%) as
compared to developing countries (15.5%). High prevalence
was for Africa (22.1%), followed by Central America and Mexico
20.4%, Northern America 11.3%, Europe 8.1% and Asia 8.0%.
HPV prevalence was highest in women younger than 34 years, with a decrease in the 35-44 year group. There was as second
peak from age 45-54 years in all regions except Asia [18]. The
predominant HPV type was HPV-16 (2.5%), followed by HPV 18
(0.9%), HPV 31 (0.7%), HPV 58 (0.6%) and HPV 52 (0.6%).

The present study shows that HPV 16 was with lower
seroprevalence in pregnant women (2.9%) as compared to nonpregnant
women (5.9%), but the difference was not statistically
significant. However, HPV 18 seroprevalence was higher in
pregnant (2.9%) than in non-pregnant (0%) women, but the
difference was not statistically significant. In contrast, Salcedo, et
al. [33] in Brazil found that HPV infection was higher in pregnant
as compared to non-pregnant. Other study by Liu, et al. [34] in
2014 conducted on the frequency of HPV among 13640 pregnant
and non pregnant women reported that frequency of HPV
infection was 16 – 82% in pregnant and 12-25% in non pregnant.
Immunological or hormonal changes may alter the prevalence of
HPV and its clearance during pregnancy [35]. Some researchers
reported decreased clearance of high-risk HPV types in the first
two trimesters of pregnancy, contributing to a high prevalence of HPV during pregnancy [36]. The prevalence of HPV DNA
among pregnant women that we observed (5.7%; 5 cases /88
samples) was lower than that in general female population of
China (13.8%) [37] and BOH cases in Iran [28,29]. Other study in
China not found a significant prevalence difference between the
prevalence of HPV DNA and the pregnancy status [38].

The vertical transmission of HPV from mother to neonates
represents a health problem with impact on child health. In 5.2%
of neonates born to healthy women were infected with HPV and
was associated with detection of HPV in mothers during any
of the pregnancy trimester [39]. Thus vaccination is the best
health care approach for the prevention and control of HPV
infections. The two commercially available HPV vaccines are
good in induction of antibody that was 7 fold higher than natural
infection for at least twenty years post vaccination [40].